Transcript Lecture 5 (2/03/10) "Protein Folding"

Short Announcements
1st Quiz today
Homework #2 on web. Due next Monday.
Chpt 2 Reading Due next Wednesday (NOT Monday)
Today’s Lecture: Protein Folding
Quiz #1 (covering Chpt 1, ECB)
1. What are the three major classes of filaments that make up the
Microtubule, actin filaments and intermediate filaments
cytoskeleton?__________________________________________
Plasma membrane
2. All cells are enclosed by a _________________that
separates the
inside of the cell from the environment.
DNA
3. All cells contain _____as
a store of genetic information and use it to
proteins
guide the synthesis of__________.
bacteria, archea, eukarya/eukaryotes
4. A) List the 3 kingdoms of life. ____________________________
eukaryotes
B) You are a member of which kingdom? __________________
5. The presence of this organelle is the most striking difference between
nucleus, membrane bound organelles
prokaryotic and eukaryotic organisms. _______________________
mitochondria
6. The ______________is
the organelle most responsible for energy
production in a eukaryotic cell.
The Protein (Free)
Energy Landscape
Largely from Martin Gruebele, Chemistry, Physics UIUC
A typical protein folding equilibrium
constant Keq ≈ 1000 means a protein
is unfolded for 100 sec/day!
A+B  A-B Keq= [A-B]/[A][B]

kf
 Afolded
kuf

Aunfolded
day
Hydrophobic regions
become exposed.
Become ubiquinated.
Reused aa in
proteasomes.
Keq= [Afolded]/[A] unfolded
=
kf/ kuf
Not nearly enough chaperones to help re-fold.
Tend to do this by itself. 20-60% are natively
unfolded– bind to negatively charged substrate
and then folds.
50-100AA
How does a Protein go from unfolded to folded
a) at all; b) in 1 msec; c)with no chaperones

Unfolded
Inactive

Folded
Active
Hans Frauenfelder,
founder of biological
physics.
Main driving force : 1) Shield hydrophobic (black spheres) residues/a.a. from solvent/
water; 2) Formation of intramolecular hydrogen bonds.
Active areas: 4 centuries on it and still not solved!
Difficulty relating to experimental observations.
In a crowded cell, chaperones are needed,
but take a protein assembly under dilute conditions,
they fold fine.
Energy and Free Energy Landscapes
Core and surface

solvent
solvent
solvent
solvent
Amino acid represented as beads
 Black
bead: hydrophobic (H)
 White bead: hydrophilic (P)
solvent
solvent
solvent
solvent
solvent
solvent
solvent
(shown: a configuration
with favorable E = <H>)

Bonds represented by straight lines

H-H (= -1000J =1/3 kT) and P-P (= -250J) bonds
favorable Peptides don’t fold because they have too few H-H and P-P to
fold stably.
H-H go inside; P-P on outside/solvent exposed
Based on work by N. Go M. Levitt, K. A. Dill, Shakhnovich/Karplus
Protein Example

6-mer

2 hydrophobic AA

4 hydrophilic AA
Chirality in Amino acids
Although most amino acids
can exist in both left and
right handed forms, Life on
Earth is made of left handed
amino acids, almost
exlusively. Why? Not really
known. Meteorites have lefthanded aa.
http://en.wikipedia.org/wiki/File:Chi
rality_with_hands.jpg


To avoid issues with chirality, all molecules are
made so that the first two amino acids go
upwards.
Also, the first kink always goes to the right.
Rotation Rules

2-D model - no
rotations allowed.

Molecules are only allowed to change by a
single 90˚ “kink” per
time step.
Allowed kinetics– one moves only 90 degrees. Kinetic
moves by diffusion.
The Journey
Direct folding!
Direct folding!
A trap!
Entropy
S  Entropy  kB ln W
Conformation Analysis
E
Reaction
Coordinate
x
0
0.33
-0.5 kJ
Kinetic
traps
No none example s where
there are multiple states.
0.66
1
Only nearest neighbors that count
Molecular Dynamics has actually taken over
This is the folding funnel:
Entropy
E
k ln14
k ln1 = 0
Entropy : horizonal scale
Entropy vs. Energy
correlated monotonic function
Entropy
Ln 14
Ln 1
-1500
-1000
Energy (kJ)
-500
0
Entropy
Entropy vs. Reaction
Coordinate
0
0.33
0.66
Reaction Coordinate
1.0
0.99
Free Energy
G is almost always flat.E goes up, S
also goes up. They compensate
G(x) = H(x) - TS(x) ≈ E(x) - TS(x)
(if compressibility is neglected so H ≈ E)
Free Energy Analysis (200K)
0
Free Energy (G)
Downhill folding (but in reality, at 200K,
nothing moves)
0
0.33
Reaction Coordinate
x
0.66
1.0
Free Energy (G)
Free Energy Analysis (298K)
Downhill folder
0
0.33
0.66
Reaction Coordinate
1.0
0.99
Free Energy Analysis (360 K)
Free Energy (G)
This is likely the equilibrium of 50:50 where
they are interconverting and equally stable.
Two state folder
Unfolded state—has some
structure
0
0.33
0.66
Reaction Coordinate
1.0
0.99
Free Energy Analysis (2000K)
Free Energy
Downhill unfolder
0
0.33
0.66
Reaction Coordinate
1.0
0.99
Energy Funnel and Free Energy Surface
Wolynes
Bryngelson
Onuchic
Luthey-Schulten
Dill
Thirumalai
Enthalpy
Config.
entropy
Free energy
DG>0
DS<0
-1
DG = DH - T DS
0
x
1
Free energy
DH<0
DG<0
-1
0
x
1
Amyloid Fibers…involved in Alzheimers
There is a lower energy state which is
Protein amyloid fibers are often found to
fibers—e.g. ameloid fibers– mutliple states!
have a β-pleated sheet structure
regardless of their sequence, leading some
to believe that it is the molecule's
misfolding that leads to aggregation.
http://www.informaworld.com/smpp/content~content=a7
79685983~db=medi~order=page
Enzymes act on the APP (Amyloid precursor protein) and cut it into fragments of protein, one
of which is called beta-amyloid and its crucial in the formation of senile plaques in Alzheimer
Enzymes act on the APP (Amyloid precursor protein) and cut it into fragments of protein, one
of which is called beta-amyloid and its crucial in the formation of senile plaques in
AlzheimerEnzymes act on the APP (Amyloid precursor protein) and cut it into fragments of
protein, one of which is called beta-amyloid and its crucial in the formation of senile plaques in
Alzheimer
Summary of Protein Folding
Proteins can fold.
Don’t need chaperones.
ΔG is always about zero. Therefore can fold fast.
Kinetics – fast cause not huge barriers
Class evaluation
1. What was the most interesting thing you learned in class today?
2. What are you confused about?
3. Related to today’s subject, what would you like to know more about?
4. Any helpful comments.
Answer, and turn in at the end of class.